RXR homodimer formation

ABSTRACT

The invention provides a method of screening a substance for the ability to effect the formation of a retinoid X receptor homodimer comprising combining the substance and a solution containing retinoid X receptors and determining the presence of homodimer formation. Also provided is a method of screening a substance for an effect on a retinoid X receptor homodimer&#39;s ability to bind DNA comprising combining the substance with the homodimer and determining the effect of the compound on the homodimer&#39;s ability to bind DNA. A method of inhibiting an activity of a retinoid X receptor heterodimer comprising increasing the formation of a retinoid X receptor homodimer, thereby preventing the retinoid X receptor from forming a heterodimer and preventing the resulting heterodimer activity is also provided. A method of inhibiting an activity of a retinoid X receptor homodimer is also provided. In addition, a method of screening a response element for binding with a retinoid X receptor homodimer is provided. Finally, the invention provides methods of activating retinoid X receptor homodimer formation.

This invention was made with government support under Grant NumbersCA51993 and CA50676 from the National Institutes of Health. The U.S.Government may have certain rights in this invention.

This application is a continuation-in-part of U.S. Ser. No. 07/901,719,filed Jun. 16, 1992, now abandoned, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The vitamin A metabolite all-trans-retinoic acid (RA) and its naturaland synthetic derivatives (retinoids) exert a broad range of biologicaleffects¹,2. Clinically, retinoids are important therapeutics in thetreatment of skin diseases and cancers³⁻⁶. Understanding how themultitude of retinoid actions can be mediated at the molecular level hasbeen greatly enhanced by the cloning and characterization of specificnuclear receptors, the retinoic acid receptors (RARs)⁷⁻¹² and theretinoid X receptors (RXRs)¹³⁻¹⁷. RARs and RXRs are part of thesteroid/thyroid hormone receptor superfamily¹⁸,9. Both types ofreceptors are encoded by three distinct genes, α, β, and γ, from which,in the case of RARs, multiple isoforms can be generated²⁰⁻²².Interestingly, while RARs are specific to vertebrates, the RXRs havebeen well conserved from Drosophila to man¹⁷,23. Despite theconsiderable advances in the understanding of the molecular mechanismsof retinoid receptor action in recent years, a central question ofwhether distinct molecular pathways for naturally occurring retinoidsexist has not yet been answered. The recent observation that the RAstereoisomer 9-cis-RA binds with high affinity to RXRs²³,24 suggested aretinoid response pathway distinct from that of all-trans-RA. However,it was almost simultaneously discovered by several laboratories thatRARs require interaction with auxiliary receptors for effective DNAbinding and function and that RXRs are such auxiliaryreceptors¹⁵,16,26-29. Hence, RARs appear to function effectively only asheterodimeric RAR/RXR complexes, or in combination with comparableauxiliary proteins that still need to be identified. Similarly, RXRswere shown to require RARs, thyroid hormone receptors (TRs), or VitaminD₃ receptors (VDRs) for effective DNA binding¹⁵,16,26-29. Thus, fromthese DNA binding studies, RXRs appeared to be able to functionpredominantly if not exclusively as auxiliary receptors, thereby playinga crucial role in generating a high degree of diversity and specificityof transcriptional controls and mediating the highly pleiotropic effectsof different hormones by increasing DNA affinity and specificity for atleast 3 different classes of ligand-activated receptors.

Contrary to these findings, the present invention provides that RXRsform homodimers. The invention provides that these homodimerseffectively bind to specific response elements in the absence ofauxiliary receptors and their DNA binding specificity is distinct fromthat of the RXR containing heterodimers. The invention demonstrates anovel mechanism for retinoid action by which a ligand induced-homodimermediates a distinct retinoid response pathway. Additionally, ligands areprovided which selectively activate RXR homodimer formation.

SUMMARY OF THE INVENTION

The invention provides a method of screening a substance for the abilityto affect the formation of a retinoid X receptor homodimer comprisingcombining the substance and a solution containing retinoid X receptorsand determining the presence of homodimer formation. Also provided is amethod of screening a substance for an effect on a retinoid X receptorhomodimer's ability to bind DNA comprising combining the substance withthe homodimer and determining the effect of the compound on thehomodimer's ability to bind DNA. A method of inhibiting an activity of aretinoid X receptor heterodimer comprising increasing the formation of aretinoid X receptor homodimer, thereby preventing the retinoid Xreceptor from forming a heterodimer and preventing the resultingheterodimer activity is also provided. A method of inhibiting anactivity of a retinoid X receptor homodimer is also provided. A methodof determining an increased probability of a pathology associated withretinoid X receptor homodimer formation and treating such pathology arefurther provided. In addition, a method of screening a response elementfor binding with a retinoid X receptor homodimer is provided. Finally,the invention provides methods of activating retinoid X receptorhomodimer formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows 9-cis-retinoic acid induces RXR homodimer bindingon TREpal.

(a) In vitro synthesized RXRa was incubated either with (+) or without(-) indicated hormones (10⁻⁷ M 9-cis-RA; 10⁻⁶ M RA; 10⁻⁶ M T₃) in thepresence or absence of in vitro synthesized TRα or RARβ for 30 min atroom temperature. After this preincubation, the reaction mixtures wereanalyzed by gel retardation assay using ³² P-labeled TREpal as probe.Lane 1 represents the nonspecific binding of unprogrammed reticulocytelysate. Open triangles indicate the nonspecific complex observed withunprogrammed reticulocyte lysate. Solid triangles indicate the specificTRα-RXRα heterodimer binding. Arrows indicate specific RXRα homodimerbinding. The RXRα/RARβ heterodimer migrates at the same position as theRXRα homodimer. For comparison, the effect of 9-cis-RA on RARβ bindingis shown.

(b) To determine that 9-cis-RA induced DNA binding complex contains RXRαprotein, Flag-RXRα (F-RXR), an RXRα derivative that contains aneight-amino-acid epitope (Flag) at its amino terminal end which can berecognized by a specific anti-Flag monoclonal antibody, was constructed.In vitro synthesized F-RXRα was incubated either with (+) or without (-)10⁻⁷ M 9-cis-RA in the presence of either specific anti-Flag antibody(αF) or nonspecific preimmune serum (NI) for 30 min at room temperature.The effect of anti-Flag antibody on F-RXRα binding in the presence of9-cis-RA was then analyzed by gel retardation assay using ³² P-labeledTREpal as a probe. Lane 1 represents the nonspecific binding ofunprogrammed reticulocyte lysate (open triangles). Arrows indicate thespecific F-RXRα homodimer and RAR-RXR heterodimer binding. Diamondsindicate the anti-Flag antibody up-shifted F-RXR homodimer.

FIGS. 2A-2C show the characterization of 9-cis-RA induced RXR homodimeron TREpal.

(a) Cooperative binding of 9-cis-RA induced RXRα homodimer. Formation ofRXR-DNA complex at different receptor concentrations in the absence orpresence of 10⁻⁷ M 9-cis-RA was analyzed by gel retardation assay usinglabeled TREpal as probe. Open triangle indicates the nonspecific bindingof an unprogrammed reticulocyte lysate. Arrows indicate the specific RXRbinding complex.

(b) Quantitation of the RXR binding complex at different receptorconcentrations in the presence of 9-cis-RA. Gel slices containing RXRbinding complex in the presence of 9-cis-RA shown in FIG. 2(a) wereexcised and counted in a scintillation counter and plotted.

(c) 9-cis-RA concentration-dependent binding of RXR homodimer on TREpal.Equal amounts of in vitro synthesized RXR protein were incubated withindicated concentrations of 9-cis-RA. The reaction mixtures were thenanalyzed by gel retardation assay using labeled TREpal as a probe. Opentriangles indicate the nonspecific binding of unprogrammed reticulocytelysate. Arrows indicate the specific RXR binding complex.

FIGS. 3A and 3B show 9-cis-RA induces RXR homodimer binding onRXR-specific response elements.

(a) Nuclear receptor binding elements used in this study. Theseoligonucleotides were synthesized with appropriate restriction sites atboth ends as indicated by the small letters. Sequences that are closelyrelated to the AGG/TTCA motif are indicated by arrows.

(b) The effect of 9-cis-RA on RXR homodimer binding of ApoAI-RARE orCRBPII-RARE was analyzed essentially as described in FIG. 1a. Lane 1represents the nonspecific binding of unprogrammed reticulocyte lysate,which are indicated by the open triangles. Solid triangles indicate theRAR/RXR heterodimer complex. Arrows indicate the specific RXR bindingcomplex.

FIGS. 4A-4C show response element specific binding of RXR homodimer. Theeffect of 9-cis-RA on RXR binding on RA specific response elements (a),T₃ specific response elements (b), or estrogen specific response element(c) was analyzed by gel retardation assays as described in FIG. 1a. Forcomparison, the binding of RXR/RAR heterodimer (a), RXR/TR heterodimer(b) or estrogen receptor (c) is shown. Open triangles indicate thenonspecific binding of unprogrammed reticulocyte lysate. Solid triangleindicates the RAR/RXR heterodimer complex (a), TR/RXR heterodimercomplex (b) or ER complexes (c).

FIG. 5 shows RXR homodimerization occurs in solution. ³⁵ S-labeled invitro synthesized RXRα proteins were incubated with partially purifiedbacterially expressed Flag-RXR (F-RXR) (+) or similarly preparedglutathione transferase control protein (-) either in the presence orabsence of response elements or chemical crosslinker DSP as indicated.After incubation, either anti-Flag antibody (F) or nonspecific preimmuneserum (NI) was added. 10⁻⁷ M 9-cis-RA was maintained during workingprocess. The immune complexes were washed in the presence of 10⁻⁷M-cis-RA, boiled in SDS sample buffer and separated on a 10% SDS-PAGE.The ³⁵ S-labeled in vitro synthesized RXRα protein is shown in the rightpanel.

FIGS. 6A-6D show transcriptional activation of RXR and RARα: RXRheterodimers by 9-cis-RA on natural response elements. CV-1 cells werecotransfected with 100 ng of the reporter plasmids (a) TREpal-tk-CAT (b)βRARE-tk-CAT (c) ApoAI-RARE-tk-CAT and (d) CRBPI-RARE-tk-CAT and 5 ng ofempty pECE expression vector, pECE-RXRα, pECE RARα or combination ofboth as indicated. Transfected cells were treated with no hormone (openbars), 10⁻⁷ M RA (shadowed bars) or 10⁻⁷ M 9-cis-RA (dark shadowedbars). The results of a representative experiment performed in duplicateare shown.

FIGS. 7A and 7B show RXRα-dependent transactivation of reporterconstructs (a) TREpal-tk-CAT (10) or (b) CRBPII-tk-CAT (10) by 9-cis-RAor retinoids SR11203, SR11217, SR11234, SR11235, SR11236, and SR11237.Results of a representative experiment carried out four times are shown.In four independent experiments, induction profiles did not varysignificantly. CAT activity was normalized for transfection andharvesting efficiency by measuring the enzymatic activity derived fromthe cotransfected β-galactosidase expression plasmid (pCH110,Pharmacia).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of screening a substance for the abilityto affect the formation of an RXR homodimer comprising combining thesubstance and a solution containing RXRs and determining the presence ofa homodimer formation. The presence of homodimer formation can, forexample, be determined by detecting the activation of transcription bythe RXR homodimer or by coprecipitation. The affect can be the inductionof homodimer formation, for example, an activity similar to thatactivated by 9-cis-RA or an activity which selectively activateshomodimer formation over heterodimer formation. By "selectivelyactivates" is meant a compound which activates homodimer formation butdoes not significantly activate heterodimers. The affect can also be theinhibition of homodimer formation. Examples of inhibition include asubstance which competes for 9-cis-RA binding to the receptor but itselfdoes not activate or induce dimerization or which binds 9-cis-RA toblock its activity. Such screening of substances is routinely carriedout given the subject discovery of homodimer formation. In particularassays set forth below can generally be used for screening by merelysubstituting the substance of interest for 9-cis-RA. A good startingpoint for screening such "substances" is the activity of 9-cis-RAdescribed herein. The substituents on 9-cis-RA can be varied to make9-cis-RA analogs and screened in the method to determine any increase ordecrease in homodimer formation. However, any substance can be screenedin this assay to determine any affect on homodimer formation. Suchcompounds can then be used to promote homodimer formation and genetranscription in a cell. A cell as used herein includes cells foundeither in vitro or in vivo. Thus, the compounds can be administered to ahuman subject to effect RXR homodimer formation and promotetranscription of a gene activated by an RXR homodimer. Such compoundsare set forth in the Examples.

The data set forth herein utilizes RXRα. However, given the highhomology between RXRα, β and γ, each protein should form homodimers andhave the activity described for RXRα homodimers. Relatedly, homodimerscan form between different RXRs. For example, homodimers can formbetween RXRα and RXRβ or between RXRβ and RXRγ or between RXRα and RXRγ.The activity of these homodimers can be confirmed using the methods setforth herein.

The invention also provides a method of screening a substance for aneffect on an RXR homodimer's ability to bind DNA comprising combiningthe substance with the homodimer and determining the effect of thecompound on the homodimer's ability to bind DNA. For example, compoundswhich might bind the homodimer or bind the DNA response elementrecognized by an RXR homodimer can be screened in this method.

The invention further provides a method of inhibiting an activity of anRXR-containing heterodimer comprising increasing the formation of an RXRhomodimer, thereby preventing the RXR from forming a heterodimer andpreventing the resulting heterodimer activity. The activity can be anyactivity but is generally the activation or repression of transcription.The activity can be blocked, for example, by utilizing RXRs to formhomodimers which otherwise would be available to form heterodimers.Since the number of heterodimers are decreased, the activity of theheterodimers is decreased. In one example, the RXR heterodimer iscomprised of thyroid hormone receptor and RXR. The activity of theRXR/TR heterodimer was decreased. Other heterodimers can be tested usingstandard methods given the teaching set forth herein.

The invention also provides a method of inhibiting an activity of an RXRreceptor homodimer comprising preventing the formation of the RXRhomodimer. Such inhibition can be obtained, for example, by inhibiting9-cis-RA or the transcription or activation by 9-cis-RA. The activityinhibited is generally the activation or repression of transcription.

The invention also provides a method of inhibiting an activity of an RXRhomodimer comprising preventing the binding of the RXR homodimer to itsresponse element. For example, the activity of a receptor which competesfor the same response element can be promoted. In general, the activitywhich is inhibited is the activation or repression of transcription.

The invention still further provides a method of determining anincreased probability of a pathology associated with RXR homodimerformation comprising detecting a modulation of RXR homodimer formationin the subject when compared to a normal subject. The modulation can bean increase or a decrease in homodimer formation. Such a modulation canresult, for example, from a mutated RXR. The decrease can be detected byan assay for the homodimers in a sample or by detecting mutations knownto decrease homodimer formation.

Relatedly, the invention provides a method of treating a pathologyassociated with RXR homodimer formation in a subject comprisingmodulating homodimer formation in the subject. The modulation can be anincrease or a decrease depending on the pathology. Such an increase canbe accomplished, for example, utilizing compounds which promote RXRtranscription. The pathology can be associated with the skin, e.g., acneand psoriasis. In addition, the pathology can be a cancer. The exactamount of such compounds required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease that is being treated, the particular compoundused, its mode of administration, and the like. Thus, it is not possibleto specify an exact activity promoting amount. However, an appropriateamount may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein.

The invention also provides a purified RXR homodimer. By "purified" ismeant free of at least some of the cellular components associated withRXR homodimers in a natural environment.

Finally, the invention provides a method of screening a response elementfor binding with a RXR homodimer comprising combining the responseelement with the RXR homodimer and detecting the presence of binding.The presence of binding can be determined by a number of standardmethods. In one method, binding is detected by the transcriptionalactivation of a marker which is operably linked to the response element.By "operably linked" is meant the marker can be transcribed in thepresence of the transcriptional activator.

The invention grows out of our study of the effects of the naturalvitamin A derivative 9-cis-RA on retinoid receptor DNA binding andtranscriptional activation. In contrast to all-trans-RA, the 9-cisanalog dramatically enhances RXRα binding at 10⁻⁹ to 10⁻⁸ Mconcentrations to several RXR-specific RAREs but not to natural TREs orthe ERE. The effect is specific to RXR since 9-cis-RA did not inducebinding of RARα, β or γ to response elements (FIGS. 1A-1B, FIG. 3B).Judging from the migration pattern in the gel shift assays, we assumethat 9-cis-RA induces homodimer formation, although a larger complexcontaining an RXR trimer or tetramet can occur (particularly in the caseof the CRBPII-RARE). Such trimer or tetraruer formation can be testedusing the methods set forth herein.

RXRα homodimers exert response element specificity distinct fromheterodimers. The rCRBPI response element did not interact with RXRhomodimers, while the CRBPII response element, the only natural RAREidentified so far that contains perfect repeats, was a strong binder of9-cis-RA induced RXRα homodimers. It has been shown previously that thisresponse element is well activated by RXRα²⁴,30. Although this responseelement is also bound effectively by the RXRα-RARα heterodimer (FIG.3B)²⁷,29, the heterodimer appears to have a repressor function³⁰.

The results obtained with the transcriptional activation studies agreewell with the DNA binding studies although CV-1 cells, like all othermammalian tissue culture cells tested, contain endogenous retinoidreceptors that can partially obscure effects. Nonetheless, responseelements that strongly bind 9-cis-RA-RXRα homodimers also respondedstrongly to cotransfected RXRα in the presence of 9-cis-RA, whereasresponse elements that did not bind well to RXRα homodimers, like therCRBPI-RARE or the MHC-TRE, could not be activated by RXRα alone.

It is generally believed that the dimerization--homodimerization orheterodimerization--of nuclear hormone receptors is critical for highaffinity interaction of the receptors with their cognate responseelements. RXRs exist mainly as monomer in solution¹⁶ and require highconcentrations or the presence of RARs, TRs or VDR to display effectiveDNA binding activity¹³,15,16,26-30. The observation of the enhancedcooperative RXR DNA binding activity in the presence of 9-cis-RAdemonstrates that 9-cis-RA induced the formation of RXR homodimers whichhave an increased affinity for DNA. Thus, binding of 9-cis-RA to RXR caninduce a conformational change, which allows homodimerization to occur.It is interesting that although 9-cis-RA and RA can bind to RAR,²⁴,25they do not induce RAR homodimer formation.

RXRα homodimer formation can occur in solution in the absence of DNA.Thus, when 9-cis-RA becomes available to cells, the equilibrium betweenmonomeric and dimeric receptors is changed and an additional species,the RXR homodimer can be formed, allowing for novel response pathways.The concept of ligand-induced homodimer binding as observed by in vitrogel shift assay has not been previously observed for nuclear receptorswith the exception of a mutated estrogen receptor (ER-val-400)⁴⁴,45.

For the related TRs, a ligand effect has been reported on homodimerbinding, ⁴⁶,47 however the ligand (T3) reduced homodimer responseelement interaction. Since the carboxy terminal half of TRs and RARsencodes both ligand as well as dimerization functions³⁶,46,48, thestrong effect of the ligand on dimerization as observed here is notcompletely surprising. However, the specificity of the effect is quitedramatic since only homodimer but not heterodimer formation appears tobe affected. An overall picture emerges where the carboxy terminalregion of a receptor through its intermixed domains (that also includesa transcriptional activation region)⁴⁹ allows for multiple activities ofindividual receptors that may also include interactions with otherregulatory proteins⁵⁰.

The data presented in this application clearly demonstrate the centralrole of the RXRs, having dual functions that allow them to act asauxiliary receptors for three classes of hormone receptors, the RARs,TRs and VDRs through heterodimerization. The two functions ofRXR--homodimerization and heterodimerization--represent two distincttranscriptional regulatory controls that can be expected to affectdistinct physiological processes. Thus, 9-cis-RA can have therapeuticproperties distinct from that of all-trans-RA.

EXAMPLE I Identification of Homodimer Formation

9-cis-RA Induces RXR Homodimer Binding on the TREpal

Although RXRs have been shown to bind RA response elements (RAREs) whenused at high concentrations,²⁸,30,31 more recent investigations revealedthat RXR exists mainly as monomers in solution¹⁰ and that effective DNAinteraction requires heterodimer formation with RARs or TRs orVDR¹⁵,16,26-29. Binding of the heterodimers to a variety of responseelements was found to be ligand independent³². The newly discoverednatural RA isomer, 9-cis-RA, has been reported to be an effectiveactivator of RXRs in Drosophila Schneider cells that are known tocontain neither RAR nor TRs¹⁷,24. If 9-cis-RA is indeed a true ligandfor RXRs, one might expect that this ligand modulates RXR responseelement interaction. We therefore investigated the effect of 9-cis-RA onRXRα binding to the palindromic TRE (TREpal), an RXR responsiveelement¹⁴, in the absence and presence of coreceptors (TRs and RARs).

The cloning of the receptor cDNAs for RXRα, RARβ, or TRα into thepBluescript (Stratagene, San Diego, Calif.) have been describedpreviously²⁶. Flag-RXRα was constructed as described previously³³ byligation of a double-stranded oligonucleotide containing an ATG codonand a DNA sequence encoding Flag (Arg-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) [SEQID NO:1] to the N-terminus of RXRα. The fusion product was then clonedinto pBluescript. The synthesis of receptor proteins using in vitrotranscription/translation system and gel retardation assays usingsynthesized receptor proteins and the double-stranded TREpal³⁴ were asdescribed³³. 9-cis-RA (m.p. 184°-187° C.) was prepared from9-cis-retinal by a two-step sequence of MnO₂ oxidation in the presenceof HOAc-MeOH³⁵ to give the methyl ester (69%) followed by hydrolysis(80%) in 0.5N KOH in 25% aq. MeOH and crystallization (MeOH); HPLC(Novapak C₁₈, 32% MeCN, 27% MeCH, 16% I-PrOH, 24% H₂ O, 1% HOAc, 1.9mL/min, 260 nM) t_(R) 15.4 min (100%). To analyze the effect ofhormones, the receptor proteins were incubated with appropriateconcentrations of hormone at room temperature for 30 min beforeperforming the DNA binding assay. When anti-Flag antibody (Immunex,Seattle, Wash.) was used, 1 μl of the antiserum was incubated withreceptor protein for another 30 min at room temperature beforeperforming the DNA binding assay.

While in the absence of ligand, RXR alone did not bind effectively tothe TREpal and required TR or RAR for response element interaction,binding of RXR was dramatically increased in the presence of 9-cis-RA(FIG. 1a) and did not require TRs or RARs. The RXR-specific bandobserved in the presence of 9-cis-RA was as prominent as the bandsobtained with the heterodimeric TR-RXR and RAR-RXR complexes. The RXRcomplex migrated more slowly than the TR-RXR complex at a position verysimilar to that of the RAR-RXR heterodimer-TREpal complex. These datatherefore demonstrate that 9-cis-RA induces RXR homodimer formation. Dueto migration of the RAR-RXR complex at the same position as the 9-cis-RAinduced RXR homodimer complex, we were unable to determine in thisexperiment whether both complexes were formed. Remarkably, all-trans-RAwhen added at a concentration of 10⁻⁶ M, also induced to some degreeRXRα homodimer binding whereas T₃ did not. Although the RA effect couldbe observed with several freshly prepared RA solutions, it is not clearat this point whether this homodimer formation in the presence of RA isdue to 9-cis-RA impurities in the all-trans-RA solutions used here, oris a direct effect of all-trans-RA (see below). Interestingly, althoughit was reported that 9-cis-RA is capable of binding to RAR²⁴, unlike itseffect on RXR, 9-cis-RA was not found to induce RAR homodimer binding tothe TREpal.

To provide further evidence that the observed complex in the presence of9-cis-RA indeed was an RXR complex, we performed the DNA bindingexperiment with Flag-RXR, a derivative that carries an 8 amino acidaminoterminal epitope recognized specifically by anti-Flag (αF)antibody³³. As shown in FIG. 1b, αF supershifted the 9-cis-RA inducedcomplexes, while a nonspecific antibody did not. This proves that thecomplex observed with the TREpal in the presence of 9-cis-RA indeedcontained RXR protein. To examine how dependent the 9-cis-RA inducedhomodimer formation is on the concentration of RXR protein, increasingconcentrations of in vitro translated RXR protein were mixed with thelabeled TREpal in the presence or absence of 9-cis-RA (FIG. 2a). Whenthese mixtures were analyzed by the gel retardation assay, a strongcooperative effect in homodimeric DNA binding was seen, positivelydependent on the RXRα protein concentration (FIG. 2a,b). Although slightbinding of RXR can be observed when high concentrations of RXR wereused, 9-cis-RA was required for efficient complex formation at allreceptor concentrations used. We further determined the concentrationsof 9-cis-RA required for homodimer complex formation at all receptorconcentrations used. We further determined the concentrations of9-cis-RA required for homodimer complex formation and observed asignificant effect already at 10⁻⁹ M while optimal binding was seen at10⁻⁸ M (FIG. 2c). Thus, effective RXR homodimer DNA interaction isdependent on RXR protein concentration and can occur at low levels of9-cis-RA.

9-cis-RA induced homodimeric interaction with specific response elements

RXR containing heterodimers have a highly specific interaction withvarious natural response elements in that TR-RXR heterodimers only bindstrongly to TREs but not to RAREs, whereas the opposite is true forRAR-RXR heterodimers¹⁵,32. We examined the sequence requirement of DNAbinding of 9-cis-RA induced RXR homodimer using a number of natural andsynthetic response elements (FIG. 3a).

Gel retardation assays using in vitro synthesized receptor protein aredescribed in the FIGS. 1A-1B legend. The following oligonucleotides andtheir complements were used as probes in FIGS. 3B and 4A-4C. ApoAI-RARE,a direct repeat response element with 2 bp spacer³¹,gatcAGGGCAGGGGTCAAGGGTTCAGTgatc [SEQ ID NO:2]; CRBPII-RARE, a directrepeat RXR-specific response element with 1 bp spacer³⁰,gatcCAGGTCACAGGTCACAGGTCACAGTTCAAgatc [SEQ ID NO:3]; βRARE, a directrepeat of RA response element present in RARβ promoter³⁶,37,gatctGTAGGGTTCACCGAAAGTTCACTCagatc [SEQ ID NO:4]; CRBPI-RARE, a directrepeat RA specific response element present in rat CRBPI promoter³⁸,gatccAGGTCAAAAAGTCAGgatc [SEQ ID NO:5]; MHC-TRE, a direct repeat T₃specific response element present in rat α-myosin heavy chain gene⁴⁰,gatcCTGGAGGTGACAGGAGGACAGCgatc [SEQ ID NO:6]; ME-TRE, a direct repeat T₃specific response element present in the rat malic enzyme gene⁴¹,gatcCAGGACGTTGGGGTTAGGGGAGGACAGTGGgatc [SEQ ID NO:7]; DR-4, an idealizeddirect repeat T₃ specific response element with 4 bp spacer³⁹,gatcTCAGGTCATCCTCAGGTCAgatc [SEQ ID NO.8]; DR-5, an idealized directrepeat RA specific response element with 5 bp spacer³⁹,gatcTCAGGTCATCCTCAGGTCAgatc [SEQ ID NO:9]; ERE, a perfect palindromic ERresponse element⁴², gatcTCAGGTCACTGTGACCTGAgatc [SEQ ID NO:10]. Thesequence of TREpal [SEQ ID NO:11] is shown for comparison.

ApoAI-RARE (a direct repeat response element that contains a 2 bpspacer), which has been suggested to be RXR-specific³¹, resulted in astrong RXR complex in the presence of 9-cis-RA and to a lesser degreewith RA (10⁻⁶ M). The RXR-RAR heterodimer also bound effectively to thisresponse element. Since the heterodimer complex migrated at the sameposition as RXR homodimers, the effect of 9-cis-RA on RXR-RARheterodimers cannot be clearly determined. RAR homodimer binding was notinduced by 9-cis-RA (FIG. 3b). When we investigated 9-cis-RA induced RXRbinding to another RXR responsive element³⁰, the CRBPII-RARE, weobserved a complex that migrated more slowly than the heterodimer (inthe absence of ligand), while in the presence of 9-cis-RA and RAR, bothhomodimer and heterodimer binding appeared to be reduced in theirintensity (FIG. 3B); a similar effect was seen at high RA concentrationsor when both 9-cis-RA and RA were present. 9-cis-RA also induced RXRinteraction with the βRARE (FIG. 4a), the RAR response element from thehuman RARβ promoter³⁶,37 that contains a 5 bp spacer. However, the RXRhomodimer band was considerably weaker than the RAR-RXR heterodimer bandat the protein concentrations used. Interestingly, another natural RAREderived from the rat CRBPI promoter³⁸, that like the ApoAI-RARE containsa 2 bp spacer, did not show any binding of RXR in the presence of9-cis-RA (FIG. 4a), indicating that the actual sequence of the repeatedcore motif is critical for RXR homodimer binding. Similarly, theDR-5-RARE, a perfect repeat element derived from the β-RARE³⁹ did notexhibit interaction with RXR in the presence of 9-cis-RA, while itinteracted strongly with RXR when RARE was present (FIG. 4a).

To examine whether RXR homodimer binding is specific to certain RAREs,we also performed gel shift experiments with the T₃ response elementsfrom the rat α-myosin heavy chain promoter (MHC-TRE)⁴⁰, the rat malicenzyme (ME-TRE)⁴¹ and the perfect repeat DR-4³⁹. In all three cases,specific binding of RXR in the presence or absence of 9-cis-RA could notbe observed, while all three response elements bound effectively TR/RXRheterodimers (FIG. 4b), consistent with the notion that these elementsare not induced by retinoids. Similarly, the perfect pal indromic ERE⁴²also did not interact with RXR homodimers (FIG. 4c).

9-cis-RA induced homodimer formation occurs in the absence of DNA

It was reported the RXR exists mainly as monomer in solution¹⁶. Animportant question is whether 9-cis-RA induced RXR homodimers, like theRXR containing heterodimers, can form in solution in the absence of DNA.To address this question we took advantage of the Flag-RXR derivativethat can be specifically precipitated with anti-Flag antibody while RXRwild type cannot.

Flag-RXRα was cloned in frame in the expression vector pGex 2T(Pharmacia) and was expressed in bacteria using the procedure providedby the manufacturer. Protein was partially purified on a prepackedglutathione sepharose 4B column (Pharmacia) and tested for its functionby gel retardation assays and western blotting using anti-Flag antibody.Immunocoprecipitation assay was performed essentially as described²⁶.Briefly, 10 μl of ³⁵ S-labeled in vitro synthesized RXRα protein wasincubated with 5 μl (approximately 0.1 μg) of partially purifiedbacterially expressed Flag-RXRα fusion protein or similarly preparedglutathione transferase control protein in 100 μl buffer containing 10⁻⁷M 9-cis-RA, 50 mM KCl and 10% glycerol for 30 min at room temperature.When assayed in the presence of chemical cross-linker oroligonucleotides, we added 2 μl of 100 mM dithiobissuccinimidylpropionate (DSP) dissolved in DMSO or 10 ng ofoligonucleotide and continued the incubation at room temperature for 15min. The reaction mixtures were then incubated with 1 μl of anti-Flagantibody or nonspecific preimmune serum for 2 h on ice. Immune complexeswere precipitated by adding 50 μl of protein-A-sepharose slurry andmixing continuously in the cold room for 1 h. The immune complexes werewashed extensively with cold NET-N buffer (20 mM Tris, pH 8.0, 100 mMNaCl, 1 mM DTT, 0.5% NP-40) containing 10⁻⁷ M 9-cis-RA, boiled in SDSsample buffer and resolved by SDS-polyacrylamide gel electrophoresis.The gel was fixed, dried and visualized by autoradiography.

When we mixed Flag-RXR with in vitro labeled ³⁵ S-RXR protein, thelabeled RXR could be coprecipitated in the presence of anti-Flagantibody but not in the presence of nonspecific serum (FIG. 5).Coprecipitation efficiency was slightly increased in the presence of theApoAI-RARE but not in the presence of the MHC-TRE. In addition,incubation with a crosslinker (DSP) further enhanced coprecipitation ofthe labeled RXR. In all cases, specific coprecipitation was onlyobserved in the presence of 9-cis-RA. These data, therefore, give strongsupport to the assumption that 9-cis-RA induced RXR homodimer formationoccurs in solution and does not require RXR-DNA interaction.

Response element specific transcriptional activation by 9-cis-RA andRXRα

To investigate whether 9-cis-RA/RXRα homodimer response elementinteraction can be correlated with transcriptional activation of suchresponse elements by the 9-cis-RA/RXR complex, we carried out a seriesof transient transfection assays in CV-1 cells, where we cotransfectedreceptor expression vectors with CAT reporter constructs that carriedvarious response elements upstream of the tk promoter. CV-1 cells weretransiently transfected using a modified calcium phosphate precipitationprocedure as described previously⁴³. CAT activity was normalized fortransfection efficiency by measuring the enzymatic activity derived fromthe cotransfected β-galactosidase expression plasmid (pCH110,Pharmacia). The transfected cells were grown in the absence or presenceof 10⁻⁷ M 9-cis-RA or all-trans-RA.

With the TREpal containing reporter, we observed strong activation byRXRα in the presence of 9-cis-RA and little activation when RA wasadded. Activation could be further enhanced by cotransfection of RARα.In this case, however, RA also functioned as an effective activatoralthough not as efficiently as 9-cis-RA. Overall, activation by 9-cis-RAin the presence of RARα and RXRα was approximately twice as strong asseen with RXRα alone, consistent with the DNA binding data where bindingwas observed by both the heterodimer and the homodimer in the presenceof 9-cis-RA when both receptors were present (FIG. 6a). When we examinedthe βRARE (FIG. 6b), we observed that this response element was highlyactivated by endogenous CV-1 cell receptors consistent with previousobservations³⁷,42, such that further activation by low concentrations ofcotransfected receptors could not be observed. Interestingly, however,9-cis-RA was a more potent activator at 10⁻⁷ M than RA. These data thusindicate that CV-1 cells contain endogenous retinoid receptor activitythat is particularly active on the βRARE and is responsive to 9-cis-RA.

The ApoAI element containing reporter was also very effectivelyactivated by RXRα in the presence of 9-cis-RA (FIG. 6c) while RA did notinduce above the level obtained in the absence of RXRα. Similar to theTREpal, maximal activation was seen when both receptors RXRα and RARαwere cotransfected. Under these conditions RA also led to a strongactivation. In contrast, the CRBPI element, where we did not observe DNAbinding by RXR in the presence of 9-cis-RA, also was not activated byRXR and 9-cis-RA in the transient transfection studies (FIG. 6d) whileRARα alone led to significant activation that was mostly 9-cis-RAdependent. The heterodimer RARαRXRα allowed maximal activation in thepresence of 9-cis-RA. Not unexpectedly, no induction by RXRα and9-cis-RA was observed on the MHC-TRE, the ME-TRE or the ERE. These invivo analyses showed a very significant correlation to the resultsobtained with the in vitro DNA binding studies, in that strongactivation by RXRα in the presence of 9-cis-RA is only observed on theresponse elements that strongly interact with the 9-cis-RA induced RXRαhomodimer.

9-cis Retinoic acid inhibits activation by TR/RXR heterodimer

We have observed that a CAT reported gene that is activated by a thyroidhormone receptor/RXR heterodimer in the presence of thyroid hormone (T₃)can be inhibited by adding 9-cis-RA. This type of inhibition is mosteasily measured by using a transfection assay.

EXAMPLE II Identification and selection Of compounds which induce RXRactivity

We used the TREpal-tk-reporter gene in a transient transfection assayessentially as described⁵¹ to evaluate compounds for induction of RXRactivity. Briefly, CV-1 cells or Hep G2 cells were grown in DME mediumsupplemented with 10% fetal calf serum. Cells were plated at 1.0×10⁵ perwell in a 24-well plate 16-24 h before transfection. In general, 100 ngof reporter plasmid, 150 ng of β-galactosidase expression vector(pCH110, Pharmacia), and variable amounts of receptor expression vectorwere mixed with carrier DNA (pBluescript) to 1,000 ng of total DNA perwell. Chloramphenicol actyl transferase (CAT) activity was normalizedfor transfection efficiency by the corresponding β-galactosidaseactivity as previously described⁵². As shown in Example I, the TREpalrepresents a response element that is activated by both RAR/RXRheterodimers and RXR homodimers. When the RXR expression vector iscotransfected with the TREpal-tk-reporter gene into CV-1 cells,all-trans-RA does not efficiently activate the reporter, whereas9-cis-RA does. Evaluation of a series of retinoids indicated thatseveral showed activity with RXR. The pharmacophoric elements of thesestructures were then combined and further modified to produce a subsetof retinoids whose activation profiles for RXR were similar to that of9-cis-RA. Induction curves for several retinoids active with RXR areshown in FIG. 7a. Interestingly, while none of the active compoundsrevealed activity at 10⁻⁸ M, all showed activities similar to 9-cis-RAat 10⁻⁷ M. We next used a reporter gene carrying the RARE³⁰ of thecytoplasmic retinol binding protein II (CRBPII), a response element thatis only activated by RXR homodimers, but not by RAR/RXR heterodimers.The induction profiles obtained with this response element were similarto the TREpal responses (FIG. 7b). Thus, the synthetic retinoidsSR11203, SR11217, SR11234, SR11235, SR11236, and SR11237 appeared to beeffective activators of RXRα. The structures of the compounds are asfollows: ##STR1##

The activity rankings for this series of retinoids were the same forboth the TREpal and CRBPII reporter genes. The ketal SR11237 was themost active, followed by the isopropylidenyl retinoid SR11217, thehemithioketal SR11235 and the thioketal SR11234. The dithiane SR11203and dioxane SR11236 had the lowest activity. Conformational analysisindicated that these retinoids had spatial orientations of thelipophilic head and carboxyl terminus that were similar to those of9-cis-RA and that activity could be related to the length and volume ofthe substituent group (CRR') linking the tetrahydronaphthalene andphenyl ring systems.

Given the showing in Example I that 9-cis-RA specifically activates RXRαby inducing RXRα homodimer formation, we investigated theretinoid-induced RXR homodimer binding to the TREpal using a gelretardation assay. Briefly, gel retardation assays were carried outessentially as described previously³³. In vitro translated receptorreceptor protein (1 to 5 ml depending on the translation efficiency) wasincubated with the ³² P-labeled oligonucleotides in a 20-ml reactionmixture containing 10 mM Hepes buffer, pH 7.9, 50 mM KCl, 1 mM DTT, 2.5mM MgCl, 10% glycerol, and 1 mg of poly(dI-dC) at 25° C. for 20 minutes.The reaction mixture was then loaded on a 5% nondenaturingpolyacrylamide gel containing 0.5×TBE (1×TBE=0.089M Tris-borate, 0.089Mboric acid, and 0.002M EDTA).

In the absence of 9-cis-RA, RXR did not bind to this response element.Retinoids SR11217, and SR11237 induced RXR homodimer binding to theresponse element in a concentration-dependent manner. Retinoid 11203,which behaved as a weak activator in the transient transfection assays,also induced only weak RXR binding. SR11231 which did not activate theRXR homodimer was also not able to induce RXR homodimer binding. Similarresults were obtained with the CRBPII-RARE and the ApoAI-RARE. We havethus defined here a class of synthetic retinoids that activate RXRa byinducing homodimer formation and binding to DNA.

An important question was whether these RXR-active compounds, like9-cis-RA, would also activate RAR/RXR heterodimers or whether they wouldbe truly RXR selective. To analyze this, we used four different reporterconstructs carrying either the i) rat cytoplasmic retinol bindingprotein I (CRBPI) gene RARE³⁸ that is only bound and activated byRAR/RXR heterodimers; ii) the RARβ2 gene promoter RARE³⁶,37, which ismost effectively bound by heterodimers but also activated to some degreeby RXR homodimers; iii) the CRBPII-RARE, which is activated only by RXRhomodimers, and on which RAR represses RXR activity³⁰ ; iv) theapolipoprotein AI (apoAI) gene RARE³¹ that is bound and activated byRAR/RXR heterodimers as well as by RXR homodimers. The four differentreporter constructs were cotransfected with RARα, RARβ, RXRα, or withRXRα and RARα together⁵¹. The retinoids were analyzed at a concentrationof 5×10⁻⁷ M (a dose shown to yield almost full induction (FIGS. 7A-7B).

CV-1 cells were cotransfected with 100 ng reporter plasmid a)CRBPI-tk-CAT, b) βRARE-tk-CAT, c) CRBPII-tk-CAT, and d) apoAI-tk-CAT.Retinoids were applied at 5×10⁻⁷ M. Results of a representativeexperiment are shown.

The RXR-specific retinoids behaved strikingly different from 9-cis-RA(or RA) in that they only activated RXR homodimers but not RAR/RXRheterodimers. As with 9-cis-RA, both SR11217 and SR11237 were strongactivators of the CRBPII-RARE (i.e. the RARE that is significantlyactivated only by the RXR homodimer). However, in contrast to 9-cis-RA,they did not induce the CRBPI-RARE that is activated only by the RAR/RXRheterodimer. Thus, while SR11217 and SR11237 behaved very similarly to9-cis-RA on the CRBPII-RARE, they showed no response on the CRBPI-RARE,where 9-cis-RA is the optimal activator. The βRARE was slightlyactivated by SR11217 and SR11237, consistent with the relatively lowaffinity of RXR homodimers for this response element. The apoAI-RARE wasmost effectively activated by RAR/RXR heterodimers in the presence of9-cis-RA. In addition to the activity found in CV-1 cells, a significantand RXR-specific activation by retinoids SR11217 and SR11237 was alsoseen in various other cell lines, including Hep G2 cells, where aparticular high response was seen. RARα and RARβ, when cotransfectedalone, were not activated significantly by any of the syntheticretinoids on any of the response elements tested. Similar negativeresults were obtained for RARγ. RARα and β are assumed to formheterodimers with endogenous RXR-like proteins in CV-1 cells, thus theseheterodimers are also unresponsive to the synthetic retinoids.

Our data demonstrate that we have identified a novel class of retinoidsthat specifically induces RXR homodimer formation and that activates RXRhomodimers on specific response elements but not RAR/RXR heterodimers.These retinoids allow the specific activation of RXR-selective responsepathways, while not inducing RAR-dependent response pathways. Theseretinoids provide a much more restricted physiological response than RAisomers or other retinoids presently used.

EXAMPLE III ##STR2##

Methyl4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]benzoate(3):

To a suspension of aluminum chloride (1.13 g, 8.5 mmol) in 1.5 mL of1,2-dichloroethane at 0° C. under argon was added a solution of1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene 1 (1.45 g, 7.7 mmol)(Kagechika, H., et al., J. Med. Chem. 31:2182 (1988)) and4-carbomethoxybenzoyl chloride 2 (1.56 g, 7.9 mmol)(4-carbomethoxybenzoyl chloride 2 was obtained from mono-methylterephthalate, which is readily available from Aldrich, in one step(SOCl₂, DMF)) in 6 mL of 1,2-dichloroethane. The resulting solution wasbrought to room temperature and stirred thereafter for 16 h. Thereaction mixture was poured onto ice water and extracted with 40% ethylacetate/hexane. The combined organic layers were washed with saturatedaqueous NaHCO₃ and brine. The solution was dried over anhydrous MgSO₄,filtered and concentrated to afford an orange solid (4.5 g). Flashchromatography (50% dichloromethane/hexane) yielded the desired product3 as a pale yellow solid (2.07 g). Recrystallization fromdichloromethane/hexane afforded the desired product 3 as a white,crystalline solid (1.96 g, 50%): m.p. 146°-148° C.; R_(f) 0.14 (50% CH₂Cl₂ /hexane). The structure of the product was also confirmed using IR,¹ H NMR and mass spectroscopy.

EXAMPLE IV

(a.)[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-(4-carbomethoxyphenyl)]-1,3-dioxolane(8): ##STR3##

To a solution of keto-ester 3 (80 mg, 0.228) in 1 mL of benzene wasadded ethylene glycol (1 mL), 1,2-bis(trimethylsilyloxy)ethane (2 mL)and a catalytic amount of p-TsOH. The reaction mixture was heated atreflux for 4 h and then cooled to room temperature. The solution waspoured into saturated aqueous NaHCO₃ and extracted with 40% ethylacetate/hexane. The combined organic layers were dried over anhydrousMgSO₄, filtered, and concentrated to afford a solid. Flashchromatography (50% CH₂ Cl₂ /hexane) yielded the desired ketal 8 as awhite solid (0.082 g, 91%): m.p. 145°-147° C.; R_(f) 0.16 (50% CH₂ Cl₂/hexane). The structure of the product was also confirmed using IR, ¹ HNMR and mass spectroscopy.

(b.)[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-(4-carboxyphenyl)]-1,3-dioxolane(9):

To a suspension of the ester 8 (50 mg, 0.127 mmol) in 75% aqueousmethanol (2 mL) was added one pellet of potassium hydroxide (0.1 g), andthe reaction mixture was stirred at 70° C. for 1 h during which time thematerial dissolved. The solution was cooled to room temperature,acidified with 1N aqueous hydrochloric acid, and then extracted with 80%ethyl acetate/hexane. The combined organic layers were dried overanhydrous MgSO₄, filtered, and concentrated to afford a white solid 9.The structure of the product was also confirmed using IR, ¹ H NMR andmass spectroscopy.

EXAMPLE V ##STR4##

(a.) Methyl4-]1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-methyl-1-propenyl]benzoate(19):

To a suspension of isopropyltriphenylphosphonium iodide (0.35 g, 0.807mmol) in 3 mL of benzene under argon at room temperature was added a0.5M solution of potassium hexamethyldisilazide in toluene (1.8 mL, 0.89mmol), and the red solution was stirred for 5 min. A solution ofketo-ester 3 (0.169 g, 0.481 mmol) in 3 mL of benzene was added, and thered solution was heated to 110°C., while approximately 4 mL of benzenewas distilled out. After 1 h, the reaction mixture was diluted with 40%ethyl acetate/hexane and washed with saturated aqueous NaHCO₃ and brine.The organic layer was dried over anhydrous MgSO₄, filtered through aplug of silica gel, and concentrated to afford a solid. Flashchromatography (40% dichloromethane/hexane) yielded the desired product19 as a white powder (0.128 g, 71%): R_(f) 0.44 (50% CH₂ Cl₂ /hexane).The structure of the product was also confirmed using IR, ¹ H NMR andmass spectroscopy.

(b.)4-[1-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-methyl-1-propenyl]benzoicacid (20):

To a suspension of the ester 19 (0.115 g, 0.304 mmol) in 75% aqueousmethanol (3 mL) was added one pellet of potassium hydroxide (0.12 g),The mixture was stirred at 75° C. for 1 h during which time the materialdissolved. The solution was cooled to room temperature, acidified with1N aqueous hydrochloric acid, and then extracted with 80% ethylacetate/hexane. The combined organic layers were dried over anhydrousMgSO₄, filtered, and concentrated to afford the desired acid 20 as awhite powder (0.11 g, 99%): m.p. 204°-206° C. The structure of theproduct was also confirmed using IR, ¹ H NMR and mass spectroscopy.

The preceding examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

Throughout this application various publications are referenced bynumbers. Following is a complete citation to the publications. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgTyrLysAspAspAspAspLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATCAGGGCAGGGGTCAAGGGTTCAGTGATC31                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATCCAGGTCACAGGTCACAGGTCACAGTTCAAGATC37                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATCTGTAGGGTTCACCGAAAGTTCACTCAGATC34                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATCCAGGTCAAAAAGTCAGGATC24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GATCCTGGAGGTGACAGGAGGACAGCGATC30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GATCCAGGACGTTGGGGTTAGGGGAGGACAGTGGGATC38                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCTCAGGTCATCCTCAGGTCAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCTCAGGTCATCCTCAGGTCAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATCTCAGGTCACTGTGACCTGAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GATCTCAGGTCATGACCTGAGATC24                                                    __________________________________________________________________________

What is claimed is:
 1. A method of screening a substance for the abilityto selectively induce the formation of a retinoid X receptor homodimerover a retinoid X receptor heterodimer comprising:a. combining in a cellexpressing a retinoid X receptor vector the substance to be screened anda second receptor capable of forming a retinoid X receptor heterodimer;b. determining by cotransfection and expression of a response elementlinked reporter gene (1) if the substance induces a retinoid X receptorheterodimer, and (2) if the substance induces the formation of aretinoid X receptor homodimer; and c. selecting the substance whichselectively induces the formation of a retinoid X receptor homodimer. 2.The method of claim 1, wherein the heterodimer is a retinoid X receptorand retinoic acid receptor.
 3. A method of screening a substance for theability to selectively induce a retinoid X receptor homodimer over aretinoid X receptor heterodimer comprising:a. combining the retinoid Xreceptor with the substance to be screened and a second receptor capableof forming a retinoid X receptor heterodimer; b. determining byco-precipitation (1) if the substance induces the formation of aretinoid X receptor homodimer, and (2) if the substance induces aretinoid X receptor heterodimer; and c. selecting the substance whichselectively induces the formation of a retinoid X receptor homodimer. 4.A method of screening a substance for the ability to selectively inducea retinoid X receptor homodimer over a retinoid X receptor heterodimerin solution comprising:a. combining the retinoid X receptor with thesubstance to be screened and a second receptor capable of forming aretinoid X receptor heterodimer, wherein the second receptor is not aretinoid acid receptor β; b. determining by gel shift assay (1) if thesubstance induces the formation of a retinoid X receptor homodimer, and(2) if the substance induces a retinoid X receptor heterodimer; and c.selecting the substance which selectively induces the formation of aretinoid X receptor homodimer.